Control of cell growth through a temperature feedback loop

ABSTRACT

The present invention relates to the use of online biomass capacitance monitoring in cultures as a way to control the growth of cells through the use of a temperature control loop. In certain embodiments, a biomass capacitance probe is used to measure the cell density, and a predetermined growth curve is used to adjust the temperature in the culture.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.62/650,430, filed Mar. 30, 2018, which is incorporated herein byreference in its entirety.

BACKGROUND OF THE INVENTION

Understanding cell growth is critical for developing efficient androbust bioreactor processes. Due to exponential growth characteristics,small perturbations in process conditions such as inoculation density,media composition, and pH can have large impacts on cell density andprocess performance. In addition standard offline measurement techniquesfail to capture important data such as the exact cell peak time anddensity. This can make cell density based process decisions such astemperature shifting difficult to time.

There is a need in the art for mechanisms to dynamically adjust cellgrowth to precisely control the timing of cell growth from inoculationto peak.

SUMMARY OF THE INVENTION

In some embodiments, the invention is directed to a method ofcontrolling the growth of cells in a bioreactor comprising: a) measuringthe viable cell density of the cells growing in culture using a biomasscapacitance probe; and b) utilizing a temperature control loop to adjustthe growth rate of the cells.

In certain embodiments, the invention is directed to a method ofmodulating reactor temperature to control the growth of cells in abioreactor comprising: a) measuring the viable cell density of the cellsgrowing in culture using a biomass capacitance probe; and b) utilizing atemperature control loop to modulate the reactor temperature and adjustthe growth rate of the cells.

In embodiments, the invention is directed to a method of minimizing thegrowth fluctuation of cells in a bioreactor comprising: a) measuring theviable cell density of cells growing in culture using a biomasscapacitance probe; and b) utilizing a temperature control loop to adjustthe growth rate of the cells.

In some embodiments, a pre-determined growth curve is utilized to adjustthe temperature in the culture.

In embodiments, a bolus feed is added to the bioreactor daily. Incertain embodiments, the daily bolus feed is begun starting on about day1, about day 1.5, about day 2, about day 2.5, about day 3, about day3.5, about day 4, about day 5, about day 6, about day 7, about day 8,about day 9 or about day 10 after the culture is inoculated. Inparticular embodiments, the daily bolus feed volume is about 1%, about2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about9%, about 10%, about 15% or more of the initial working volume. In someembodiments, the daily bolus feed volume is about 3%, about 3.1%, about3.2%, about 3.3%, about 3.4%, about 3.5%, about 3.6%, about 3.7%, about3.8, about 3.9% or about 4% of the initial working volume. In oneembodiment, the daily bolus feed volume is about 3.6% of the initialworking volume.

In embodiments, a bolus feed is added to the bioreactor hourly. Incertain embodiments, the hourly bolus feed is begun starting on aboutday 1, about day 1.5, about day 2, about day 2.5, about day 3, about day3.5, about day 4, about day 5, about day 6, about day 7, about day 8,about day 9 or about day 10 after the culture is inoculated. In anembodiment, the hourly bolus feed is about 1/24 of the calculated volumefor a daily bolus feed. In one embodiment, the hourly bolus feed isabout 0.1%, about 0.11%, about 0.12%, about 0.13%, about 0.14%, about0.15%, about 0.16%, about 0.17%, about 0.18%, about 0.19%, about 0.2%,about 0.25%, about 0.3%, about 0.4% or about 0.5% of the initial workingvolume. In particular embodiments, the hourly bolus feed volume is about0.15% of the initial working volume.

In some embodiments, the viable cell density is plotted to a temperaturerange of about 30-40° C. In certain embodiments, the viable cell densityis plotted to a temperature range of about 31-37° C. In embodiments,daily temperature oscillations in the reactor have a peak to peakamplitude of about 10° C., about 9° C., about 8° C., about 7° C., about6° C., about 5° C., about 4° C., about 3° C., about 2° C., about 1° C.or less.

In embodiments, the cells produce a polypeptide of interest. In certainembodiments, the polypeptide of interest is an antibody. In someembodiments, the cells are mammalian cells. In particular embodiments,the cells are CHO cells.

In embodiments, the biomass capacitance probe is an INCYTE probe.

In some embodiments, the pH of the culture is about 6.5 to about 8.0,about 7.0 to about 8.0, or about 7.0 to about 7.5. In particularembodiments, the pH of the culture is about 7.1 to about 7.4.

In embodiments, the culture is inoculated with cells at a density ofabout 1×10⁵, about 2×10⁵, about 3×10⁵, about 4×10⁵, about 5×10⁵, about6×10⁵, about 7×10⁵, about 8×10⁵, about 9×10⁵, or about 10×10⁵ cells/ml.In particular embodiments, the culture is inoculated with cells at adensity of about 6×10⁵ cells/ml.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Schematic for viable cell density control.

FIG. 2: Viable cell density target.

FIG. 3: Cultures grown shifted from 36.5° C. to either 35.5° C., 34.0°C., or 32.5° C. on day 5. Perturbations correspond to daily feedingevents. Cultures are overlaid with a target line, as described byEquation 1.

FIG. 4: Biomass control with a daily feeding schedule. Oscillations inthe temperature plot align with the daily feed schedule.

FIG. 5: Biomass control with an hourly feeding schedule. The target lineis completely occluded by the biomass line.

DETAILED DESCRIPTION OF THE INVENTION Definitions of General Terms andExpressions

In order that the present disclosure may be more readily understood,certain terms are first defined. As used in this application, except asotherwise expressly provided herein, each of the following terms shallhave the meaning set forth below. Additional definitions are set forththroughout the application.

The term “and/or” where used herein is to be taken as specificdisclosure of each of the two specified features or components with orwithout the other. Thus, the term “and/or” as used in a phrase such as“A and/or B” herein is intended to include “A and B,” “A or B,” “A”(alone), and “B” (alone). Likewise, the term “and/or” as used in aphrase such as “A, B, and/or C” is intended to encompass each of thefollowing aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; Aand C; A and B; B and C; A (alone); B (alone); and C (alone).

It is understood that wherever aspects are described herein with thelanguage “comprising,” otherwise analogous aspects described in terms of“consisting of” and/or “consisting essentially of” are also provided.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure is related. For example, the ConciseDictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed.,2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed.,1999, Academic Press; and the Oxford Dictionary Of Biochemistry AndMolecular Biology, Revised, 2000, Oxford University Press, provide oneof skill with a general dictionary of many of the terms used in thisdisclosure.

Units, prefixes, and symbols are denoted in their Système Internationalde Unites (SI) accepted form. Numeric ranges are inclusive of thenumbers defining the range. The headings provided herein are notlimitations of the various aspects of the disclosure, which can be hadby reference to the specification as a whole. Accordingly, the termsdefined immediately below are more fully defined by reference to thespecification in its entirety.

The use of the alternative (e.g., “or”) should be understood to meaneither one, both, or any combination thereof of the alternatives. Asused herein, the indefinite articles “a” or “an” should be understood torefer to “one or more” of any recited or enumerated component.

The terms “about” or “comprising essentially of” refer to a value orcomposition that is within an acceptable error range for the particularvalue or composition as determined by one of ordinary skill in the art,which will depend in part on how the value or composition is measured ordetermined, i.e., the limitations of the measurement system. Forexample, “about” or “comprising essentially of” can mean within 1 ormore than 1 standard deviation per the practice in the art.Alternatively, “about” or “comprising essentially of” can mean a rangeof up to 10% or 20% (i.e., ±10% or ±20%). For example, about 3 mg caninclude any number between 2.7 mg and 3.3 mg (for 10%) or between 2.4 mgand 3.6 mg (for 20%). Furthermore, particularly with respect tobiological systems or processes, the terms can mean up to an order ofmagnitude or up to 5-fold of a value. When particular values orcompositions are provided in the application and claims, unlessotherwise stated, the meaning of “about” or “comprising essentially of”should be assumed to be within an acceptable error range for thatparticular value or composition.

As described herein, any concentration range, percentage range, ratiorange or integer range is to be understood to include the value of anyinteger within the recited range and, when appropriate, fractionsthereof (such as one tenth and one hundredth of an integer), unlessotherwise indicated.

The terms “cell culture” and “culture” include any combination of cellsand medium. The methods of the present invention contemplate, withoutlimitation, perfusion cell culture and fed-batch cell culture.

As used herein, the term “fed-batch culture” refers to a method ofculturing cells, wherein the cell culture is supplemented with freshmedium, i.e., the cells are “fed” with new medium while spent medium isnot removed. Typically, a “fed-batch” culture process is performed in abioreactor and additional components (e.g., nutritional supplements) areadded to the culture at some time after initiation of the cultureprocess. The controlled addition of nutrients directly affects thegrowth rate of the culture and allows for avoidance of the build-up ofoverflow metabolites (see, for example, Wlaschin, K. F. et al.,“Fedbatch culture and dynamic nutrient feeding,” Cell CultureEngineering, 101:43-74 (2006) and Lee, J. et al., “Control of fed-batchfermentations,” Biotechnol. Adv., 17:29-48 (1999)). A fed-batch cultureis typically terminated at some point and the cells and/or components inthe medium are harvested and optionally purified.

As used herein, the terms “inoculation”, “inoculum”, and “seeding” referto the addition of cells to starting medium to begin the culture.

As used herein, the term “cell density” refers to the number of cells ina given volume of medium. Cell density can be monitored by any techniqueknown in the art, including, but not limited to, extracting samples froma culture and analyzing the cells under a microscope, using acommercially available cell counting device or by using a commerciallyavailable suitable probe introduced into the bioreactor itself (or intoa loop through which the medium and suspended cells are passed and thenreturned to the bioreactor). In embodiments, a biomass capacitance probemeasures capacitance, which is correlated to cell density.

As used herein the terms “super high cell density” and “high celldensity” are used interchangeably and refer to a cell density of atleast about 40×10⁶ cells/mL in a bioreactor. In embodiments, super highcell density or high cell density refers to a cell density that is atleast about 40×10⁶ cells/mL, 45×10⁶ cells/mL, 50×10⁶ cells/mL, 75×10⁶cells/mL, 100×10⁶ cells/mL or higher. Known cell culture techniques mayinvolve growing cells to a “first critical level” (i.e., a point duringthe cell cycle growth phase when the cell viability may be affected bythe increased concentration of waste productions (e.g., cell growthinhibitors and toxic metabolites, e.g., lactate, ammonium, etc.) beforeperfusing the cell culture and obtaining roughly 5 to 40 millioncells/mL.

As used herein, the term “viable cell density” or “VCD” refers to thenumber of live cells present in a given volume of medium under a givenset of experimental conditions. In some embodiments, viable cell densityis used interchangeably with cell density.

As used herein, the term “cell viability” refers to the ability of cellsin culture to survive under a given set of conditions or experimentalvariations. The term as used herein also refers to that portion of cellsthat are alive at a particular time in relation to the total number ofcells (e.g., living and dead) in the culture at that time.

As used herein, the “growth phase” of a cell culture refers to the phaseduring which the viable cell density at any time point is higher than atany previous time point.

As used herein, the “production phase” of a cell culture refers to thephase during which the cells produce significant amounts of protein,which accumulates for future processing.

As used herein, the term “cell integral” refers to the overall viablecell numbers during the course of a cell growth profile.

As used herein, the term “titer” refers to the total amount of proteinproduced by a cell culture, divided by a given amount of medium volume.In essence, the term “titer” refers to a concentration and is typicallyexpressed in units of milligrams of polypeptide per liter of medium.

As used herein, the terms “media”, “cell culture media” and “culturemedia”, including grammatical variations thereof, are usedinterchangeably, and refer to the nutrient solution in which cells (forexample, animal or mammalian cells) are grown in culture. Cell culturemedia is the physiochemical, nutritional, and hormonal environment forcells and typically includes at least one or more components from thefollowing: an energy source (e.g., in the form of a carbohydrate such asglucose); essential amino acids, including the twenty basic amino acidsplus cysteine; vitamins and/or other organic compounds typicallyrequired at low concentrations; lipids or free fatty acids (e.g.,linoleic acid); and trace elements (e.g., inorganic compounds ornaturally occurring elements that are typically required at very lowconcentrations, usually in the micromolar range). Media may be solid,gelatinous, liquid, gaseous or a mixture of phases and materials.

As used herein, the term “cell”, refers to animal cells, mammaliancells, cultured cells, host cells, recombinant cells, and recombinanthost cells. Such cells are generally cell lines obtained or derived frommammalian tissues which are able to grow and survive when placed inmedia containing appropriate nutrients and/or growth factors. The cellsutilized in the methods of the present invention are generally animal ormammalian cells that can express and secrete, or that can be molecularlyengineered to express and secrete, large quantities of a particularprotein into the culture medium. In one embodiment, the protein producedby the cell can be endogenous or homologous to the cell. Alternatively,the protein is heterologous, i.e., foreign, to the cell.

The cells utilized in the methods of the present invention can be grownand maintained in any number of cell culture media, including thosewhich are known in the art or are commercially available. One ofordinary skill in the art may opt to use one or more known cell culturemedia that is selected to maximize cell growth, cell viability, and/orprotein production in a particular cultured host cell. Exemplary cellculture media include any media suitable for culturing cells that canexpress a protein of interest. In some embodiments, the media ischemically defined media.

Additionally, the cell culture media can optionally be supplemented toinclude one or more additional components, in appropriate concentrationsor amounts, as necessary or desired, and as would be known and practicedby those of ordinary skill in the art. Exemplary supplements include,but are not limited to, chemical gene selection agents, hormones andother growth factors, (e.g., insulin, transferrin, epidermal growthfactor, serum, somatotropin, pituitary extract, aprotinin); salts (e.g.,calcium, magnesium and phosphate), and buffers (e.g., HEPES(4-[2-Hydroxethyl]-1-piperazine-ethanesulfonic acid)); nucleosides andbases (e.g., adenosine, thymidine, hypoxanthine); protein andhydrolysates; antibiotics (e.g., gentamycin); cell protective agents(e.g., a Pluronic polyol (PLURONIC® F68)) and extracellular matrixproteins (e.g., fibronectin). Supplements that support the growth andmaintenance of particular cell cultures are able to be readilydetermined by those of ordinary skill in the art, such as is described,for example, by Barnes et al. (Cell, 22:649 (1980)); in Mammalian CellCulture, Mather, J. P., ed., Plenum Press, N Y (1984); and in U.S. Pat.No. 5,721,121.

As used herein, the term “bioreactor” refers to any apparatus, closedcontainer or vessel (e.g., a fermentation chamber) that is used forgrowing cell cultures. Bioreactors allow controlling various parametersduring the cell culture process including, but not limited to, thecirculation loop flow, pH, the temperature, the agitation, the gas flowrate, the feed addition, the overpressure and/or the medium perfusionrate. Bioreactors include commercially available bioreactors, classicalfermenters and cell culture perfusion systems, as well as disposablebioreactors. In some embodiments, the bioreactor is a perfusion N-1bioreactor. In other embodiments, the bioreactor is a standard batch N-1bioreactor. In further embodiments, the bioreactor is a feed N-1bioreactor. In embodiments, the bioreactor is a production bioreactor.In a particular embodiment, the bioreactor is a batch productionbioreactor. In certain embodiments, the bioreactor is a fed-batchproduction bioreactor. In some embodiments, the bioreactor is a seedbioreactor.

The bioreactor can be of any size that is useful for culturing cells ata desirable scale in accordance with a method of the invention. Forexample, a bioreactor employed in the methods of the present inventionmay be at least about 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125,130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195,200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265,270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 340,350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480,490, 500, 550, 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500,5,000, 5,500, 6,000, 6,500, 7,000, 7,500, 8,000, 8,500, 9,000, 9,500,10,000, 10,500, 11,000, 11,500, 12,0000, 13,000, 14,000, 15,000 litersor more, or any intermediate volume. The methods of the presentinvention can employ one or more bioreactors.

A suitable bioreactor may be composed of (i.e., constructed of) anymaterial that is suitable for holding cell cultures under the cultureconditions of the present invention and is conducive to cell growth andviability. For example, a bioreactor employed in the methods of thepresent invention can be made of glass, plastic or metal. However, thematerials comprising the bioreactor should not interfere with expressionor stability of the polypeptide product. Suitable bioreactors are knownin the art and commercially available.

A “biomass capacitance probe” refers to a probe that can measure viablecell density, among other capabilities. A biomass capacitance probe usescapacitance to measure the viable cells in a culture. Viable cells actas capacitors in an alternating electric field. The biomass capacitanceprobe can measure the charge from these cells, and report it.

The cell cultures encompassed by the methods of the present inventionmay be grown at any temperature appropriate for the cell type andculture conditions. In one embodiment, it is desirable to use atemperature between about 30° C. and 38° C., to enhance proteinproduction. In another embodiment, the temperature is at least about 25°C., 26° C., 27° C., 28° C., 29° C., 30° C., 31° C., 32° C., 33° C., 34°C., 35° C., 35.5° C., 36° C., 36.5° C., 37° C., 37.5° C., 38° C., 39°C., 40° C., or 41° C. or higher. It may also be desirable to usedifferent temperatures at different times during the culture.

Bioreactor and Cultivation Process

In embodiments, the invention is directed to an optimization of a cellculture process. In embodiments, the invention is directed to a processof controlling the growth of cells. In certain embodiments, theinvention is directed to a method of modulating reactor temperature tocontrol the growth of cells. In particular embodiments, the invention isdirected to a process that minimizes the growth fluctuation of cells. Inparticular embodiments, the process or method takes places inbioreactor. In some embodiments, the bioreactor is a tank bioreactor. Incertain embodiments, the bioreactor is an N-1 bioreactor. In someembodiments, the bioreactor is a perfusion N-1 bioreactor. In otherembodiments, the bioreactor is a standard batch N-1 bioreactor. Infurther embodiments, the bioreactor is a feed N-1 bioreactor. Inembodiments, the bioreactor is a production bioreactor. In a particularembodiment, the bioreactor is a batch production bioreactor. In certainembodiments, the bioreactor is a fed-batch production bioreactor. Insome embodiments, the bioreactor is a seed bioreactor. In an N-1bioreactor, there is an intense period of growth in the bioreactor priorto the transfer of the cells into the production bioreactor, and cellsgrown in an N-1 bioreactor are capable of being grown to a high celldensity. In certain embodiments, the bioreactor is about a 1 L, 5 L, 10L, 50 L, 100 L, 200 L, 300 L, or 500 L bioreactor.

In certain embodiments, the growth of cells in the bioreactor isoptimized by measurement of the viable cell density in the culture. Inparticular embodiments, the measurement of the viable cell density isused to adjust the temperature to modulate the growth rate of the cells.In particular embodiments, a temperature control loop is used tomodulate the reactor temperature.

In embodiments, the viable cell density is measured using a biomasscapacitance probe, also called an online capacitance probe or an onlinedielectric capacitance probe. In certain embodiments, the biomasscapacitance probe is the Hamilton Incyte biomass capacitance probe. Inparticular embodiments, the biomass capacitance probe is the Incyte LC.In particular embodiments, the biomass capacitance probe is the IncyteDN. In particular embodiments, the biomass capacitance probe is theIncyte HC. In other embodiments, the biomass capacitance probe is theIncyte HC. The Incyte measures online permittivity, and providesinformation only on viable cells in the reactor. The informationprovided by the Incyte can be used to correlate to viable cell density.In particular embodiments, the probe sends a continuous biomass signalto the DeltaV control system, which controls the bioreactors. In certainembodiments, a pre-determined viable cell density is programmed intoDeltaV using Equation 1. Active control is used from the time ofinoculation (t=0) until peak viable cell density (t=240).

$\begin{matrix}{{{Cell}\mspace{14mu} {Density}} = {\frac{A}{B*e^{({C*{({t - D})}})}} - e^{E}}} & {{Equation}\mspace{14mu} 1}\end{matrix}$

TABLE 1 Constants and variable definitions for Equation 1. A 41.2 B0.527 C −0.0165 D 178 E 0.0133 t time in hours

In embodiments, the biomass output translates linearly to a temperaturerange. In certain embodiments, this temperature range is about 30-40° C.In particular embodiments, the temperature range is about 31-37° C. Insome embodiments, the daily temperature oscillations in the reactor havea peak to peak amplitude of about 10° C., about 9° C., about 8° C.,about 7° C., about 6° C., about 5° C., about 4° C., about 3° C., about2° C., about 1° C. or less.

In embodiments, the pH in the bioreactor is maintained at about 6.0 toabout 8.0, about 6.5 to about 8.0, about 7.0 to about 8.0, about 6.5 toabout 7.5, about 6.6 to about 7.5, about 6.7 to about 7.5, about 6.8 toabout 7.5, about 6.9 to about 7.5, about 7.0 to about 7.5, or about 7.1to about 7.4. In certain embodiments, the pH in the perfusion bioreactoris maintained at about 6.0, about 6.1, about 6.2, about 6.3, about 6.4,about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7,about 7.8, about 7.9 or about 8.0.

In embodiments, the bioreactor is inoculated at a cell density of about1×10⁵, about 2×10⁵, about 3×10⁵, about 4×10⁵, about 5×10⁵, about 6×10⁵,about 7×10⁵, about 8×10⁵, about 9×10⁵, about 10×10⁵, about 2×10⁶, about3×10⁶, about 4×10⁶, about 5×10⁶, about 6×10⁶, about 7×10⁶, about 8×10⁶,about 9×10⁶, about 10×10⁶, about 2×10⁷, about 3×10⁷, about 4×10⁷, about5×10⁷, about 6×10⁷, about 7×10⁷, about 8×10⁷, about 9×10⁷, about 10×10⁷,cells/ml or a greater cell density. In particular embodiments, theproduction bioreactor is inoculated at a cell density of about 6×10⁵cells/ml.

In embodiments, a bolus feed is added to the bioreactor daily. Incertain embodiments, the daily bolus feed is begun starting on about day1, about day 1.5, about day 2, about day 2.5, about day 3, about day3.5, about day 4, about day 5, about day 6, about day 7, about day 8,about day 9 or about day 10 after the culture is inoculated. Inparticular embodiments, the daily bolus feed is added starting at day 3after the culture is inoculated. In certain embodiments, the daily bolusfeed volume is about 1%, about 2%, about 3%, about 4%, about 5%, about6%, about 7%, about 8%, about 9%, about 10%, about 15% or more of theinitial working volume. In particular embodiments, the daily bolus feedvolume is about 3%, about 3.1%, about 3.2%, about 3.3%, about 3.4%,about 3.5%, about 3.6%, about 3.7%, about 3.8, about 3.9% or about 4% ofthe initial working volume. In embodiments, the daily bolus feed volumeis about 3.6% of the initial working volume.

In some embodiments, a bolus feed is added to the bioreactor hourly. Incertain embodiments, the hourly bolus feed is begun starting on aboutday 1, about day 1.5, about day 2, about day 2.5, about day 3, about day3.5, about day 4, about day 5, about day 6, about day 7, about day 8,about day 9 or about day 10 after the culture is inoculated. Inparticular embodiments, the hourly feed is added starting about day 2.5after the culture is inoculated. In particular embodiments, the hourlybolus feed is about 1/24 of the calculated volume for a daily bolusfeed. In particular embodiments, the hourly bolus feed is about 0.1%,about 0.11%, about 0.12%, about 0.13%, about 0.14%, about 0.15%, about0.16%, about 0.17%, about 0.18%, about 0.19%, about 0.2%, about 0.25%,about 0.3%, about 0.4% or about 0.5% of the initial working volume. Inone embodiment, the hourly bolus feed volume is about 0.15% of theinitial working volume.

Polypeptides

Any polypeptide that is expressible in a host cell may be produced inaccordance with the present invention. The polypeptide may be expressedfrom a gene that is endogenous to the host cell, or from a gene that isintroduced into the host cell through genetic engineering. Thepolypeptide may be one that occurs in nature, or may alternatively havea sequence that was engineered or selected by the hand of man. Anengineered polypeptide may be assembled from other polypeptide segmentsthat individually occur in nature, or may include one or more segmentsthat are not naturally occurring.

Polypeptides that may desirably be expressed in accordance with thepresent invention will often be selected on the basis of an interestingbiological or chemical activity. For example, the present invention maybe employed to express any pharmaceutically or commercially relevantenzyme, receptor, antibody, hormone, regulatory factor, antigen, bindingagent, etc.

Antibodies

Given the large number of antibodies currently in use or underinvestigation as pharmaceutical or other commercial agents, productionof antibodies is of interest in accordance with the present invention.Antibodies are proteins that have the ability to specifically bind aparticular antigen. Any antibody that can be expressed in a host cellmay be used in accordance with the present invention. In an embodiment,the antibody to be expressed is a monoclonal antibody. In certainembodiments, the antibody is a polyclonal antibody.

In another embodiment, the antibody is a chimeric antibody. A chimericantibody contains amino acid fragments that are derived from more thanone organism. Chimeric antibody molecules can include, for example, anantigen binding domain from an antibody of a mouse, rat, or otherspecies, with human constant regions. A variety of approaches for makingchimeric antibodies have been described. See e.g., Morrison et al.,Proc. Natl. Acad. Sci. U.S.A. 81, 6851 (1985); Takeda et al., Nature314, 452 (1985), Cabilly et al., U.S. Pat. No. 4,816,567; Boss et al.,U.S. Pat. No. 4,816,397; Tanaguchi et al., European Patent PublicationEP171496; European Patent Publication 0173494, United Kingdom Patent GB2177096B.

In another embodiment, the antibody is human antibody derived, e.g.,through the use of ribosome-display or phage-display libraries (see,e.g., Winter et al., U.S. Pat. No. 6,291,159 and Kawasaki, U.S. Pat. No.5,658,754) or the use of xenographic species in which the nativeantibody genes are inactivated and functionally replaced with humanantibody genes, while leaving intact the other components of the nativeimmune system (see, e.g., Kucherlapati et al., U.S. Pat. No. 6,657,103).

Five human immunoglobulin classes are defined on the basis of theirheavy chain composition, and are named IgG, IgM, IgA, IgE, and IgD. TheIgG-class and IgA-class antibodies are further divided into subclasses,namely, IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2. The heavy chainsin IgG, IgA, and IgD antibodies have three constant region domains, thatare designated CH1, CH2, and CH3, and the heavy chains in IgM and IgEantibodies have four constant region domains, CH1, CH2, CH3, and CH4.

In another embodiment, the antibody is a humanized antibody. A humanizedantibody is a chimeric antibody wherein the large majority of the aminoacid residues are derived from human antibodies, thus minimizing anypotential immune reaction when delivered to a human subject. Inhumanized antibodies, amino acid residues in the complementaritydetermining regions are replaced, at least in part, with residues from anon-human species that confer a desired antigen specificity or affinity.Such altered immunoglobulin molecules can be made by any of severaltechniques known in the art, (e.g., Teng et al., Proc. Natl. Acad. Sci.USA., 80, 7308-7312 (1983); Kozbor et al., Immunology Today, 4, 7279(1983); Olsson et al., Meth. Enzymol., 92, 3-16 (1982)), and are madeaccording to the teachings of PCT Publication WO92/06193 or EP 0239400,all of which are incorporated herein by reference). Humanized antibodiescan also be commercially produced. For further reference, see Jones etal., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329(1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992), all ofwhich are incorporated herein by reference.

In still another embodiment, the monoclonal, polyclonal, chimeric, orhumanized antibodies described above may contain amino acid residuesthat do not naturally occur in any antibody in any species in nature.These foreign residues can be utilized, for example, to confer novel ormodified specificity, affinity or effector function on the monoclonal,chimeric or humanized antibody. In another embodiment, the antibodiesdescribed above may be conjugated to drugs for systemic pharmacotherapy,such as toxins, low-molecular-weight cytotoxic drugs, biologicalresponse modifiers, and radionuclides (see e.g., Kunz et al.,Calicheamicin derivative-carrier conjugates, US20040082764 A1).

Cells

Any mammalian cell or cell type susceptible to cell culture, and toexpression of polypeptides, may be utilized in accordance with thepresent invention. Non-limiting examples of mammalian cells that may beused in accordance with the present invention include BALB/c mousemyeloma line (NSO/1, ECACC No: 85110503); human retinoblasts (PER.C6(CruCell, Leiden, The Netherlands)); monkey kidney CV1 line transformedby SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293cells subcloned for growth in suspension culture, Graham et al., J. GenVirol., 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10);Chinese hamster ovary cells ±DHFR or ±GS (CHO, Urlaub and Chasin, Proc.Natl. Acad. Sci. USA, 77:4216 (1980)); mouse sertoli cells (TM4, Mather,Biol. Reprod., 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL70); African green monkey kidney cells (VERO-76, ATCC CRL-1 587); humancervical carcinoma cells (HeLa, ATCC CCL 2); canine kidney cells (MDCK,ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); humanlung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065);mouse mammary tumor (MMT 060562, ATCC CCLS 1); TRI cells (Mather et al.,Annals N.Y. Acad. Sci., 383:44-68 (1982)); MRC 5 cells; FS4 cells; and ahuman hepatoma line (Hep G2). In a particularly embodiment, the presentinvention is used in the culturing of and expression of polypeptides andproteins from CHO cell lines.

Additionally, any number of commercially and non-commercially availablehybridoma cell lines that express polypeptides or proteins may beutilized in accordance with the present invention. One skilled in theart will appreciate that hybridoma cell lines might have differentnutrition requirements and/or might require different culture conditionsfor optimal growth and polypeptide or protein expression, and will beable to modify conditions as needed.

As noted above, in many instances the cells will be selected orengineered to produce high levels of protein or polypeptide. Often,cells are genetically engineered to produce high levels of protein, forexample by introduction of a gene encoding the protein or polypeptide ofinterest and/or by introduction of control elements that regulateexpression of the gene (whether endogenous or introduced) encoding thepolypeptide of interest.

Certain polypeptides may have detrimental effects on cell growth, cellviability or some other characteristic of the cells that ultimatelylimits production of the polypeptide or protein of interest in some way.Even amongst a population of cells of one particular type engineered toexpress a specific polypeptide, variability within the cellularpopulation exists such that certain individual cells will grow betterand/or produce more polypeptide of interest. In certain embodiments ofthe present invention, the cell line is empirically selected by thepractitioner for robust growth under the particular conditions chosenfor culturing the cells. In particular embodiments, individual cellsengineered to express a particular polypeptide are chosen forlarge-scale production based on cell growth, final cell density, percentcell viability, titer of the expressed polypeptide or any combination ofthese or any other conditions deemed important by the practitioner.

Media and Culture Conditions

In some embodiments, a mammalian host cell is cultured under conditionsthat promote the production of the polypeptide of interest, anypolypeptide disclosed herein. Basal cell culture medium formulations arewell known in the art. To these basal culture medium formulations theskilled artisan will add components such as amino acids, salts, sugars,vitamins, hormones, growth factors, buffers, antibiotics, lipids, traceelements and the like, depending on the requirements of the host cellsto be cultured. The culture medium may or may not contain serum and/orprotein. Various tissue culture media, including serum-free and/ordefined culture media, are commercially available for cell culture.Tissue culture media is defined, for purposes of the invention, as amedia suitable for growth of animal cells, and, in some embodiments,mammalian cells, in in vitro cell culture. Typically, tissue culturemedia contains a buffer, salts, energy source, amino acids, vitamins andtrace essential elements. Any media capable of supporting growth of theappropriate eukaryotic cell in culture can be used; the invention isbroadly applicable to eukaryotic cells in culture, particularlymammalian cells, and the choice of media is not crucial to theinvention. Tissue culture media suitable for use in the invention arecommercially available from, e.g., ATCC (Manassas, Va.). For example,any one or combination of the following media can be used: RPMI-1640Medium, RPMI-1641 Medium, Dulbecco's Modified Eagle's Medium (DMEM),Minimum Essential Medium Eagle, F-12K Medium, Ham's F12 Medium, Iscove'sModified Dulbecco's Medium, McCoy's 5A Medium, Leibovitz's L-15 Medium,and serum-free media such as EX-CELL™ 300 Series (available from JRHBiosciences, Lenexa, Kans., USA), among others, which can be obtainedfrom the American Type Culture Collection or JRH Biosciences, as well asother vendors. When defined medium that is serum-free and/orpeptone-free is used, the medium is usually highly enriched for aminoacids and trace elements. See, for example, U.S. Pat. No. 5,122,469 toMather et al. and U.S. Pat. No. 5,633,162 to Keen et al.

In certain embodiments, cells can be grown in serum-free, protein-free,growth factor-free, and/or peptone-free media. The term “serum-free” asapplied to media includes any mammalian cell culture medium that doesnot contain serum, such as fetal bovine serum. The term “insulin-free”as applied to media includes any medium to which no exogenous insulinhas been added. By exogenous is meant, in this context, other than thatproduced by the culturing of the cells themselves. The term “IGF-1-free”as applied to media includes any medium to which no exogenousInsulin-like growth factor-1 (IGF-1) or analog (such as, for example,LongR3, [Ala31], or [Leu24][Ala31] IGF-1 analogs available from GroPepLtd. of Thebarton, South Australia) has been added. The term“growth-factor free” as applied to media includes any medium to which noexogenous growth factor (e.g., insulin, IGF-1) has been added. The term“protein-free” as applied to media includes medium free from exogenouslyadded protein, such as, for example, transferrin and the protein growthfactors IGF-1 and insulin. Protein-free media may or may not havepeptones. The term “peptone-free” as applied to media includes anymedium to which no exogenous protein hydrolysates have been added suchas, for example, animal and/or plant protein hydrolysates. Eliminatingpeptone from media has the advantages of reducing lot to lot variabilityand enhancing processing such as filtration. Chemically defined mediaare media in which every component is defined and obtained from a puresource, in certain embodiments, a non-animal source. In certainembodiments, the media is chemically defined and fully serum and proteinfree.

In some embodiments, one of the many individualized media formulationsthat have been developed to maximize cell growth, cell viability, and/orrecombinant polypeptide production in a particular cultured host cell isutilized. The methods described herein may be used in combination withcommercially available cell culture media or with a cell culture mediumthat has been individually formulated for use with a particular cellline. For example, an enriched medium that could support increasedpolypeptide production may comprise a mixture of two or more commercialmedia, such as, for instance, DMEM and Ham's F1 2 media combined inratios such as, for example, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, oreven up to 1:15 or higher. Alternatively or in addition, a medium can beenriched by the addition of nutrients, such as amino acids or peptone,and/or a medium (or most of its components with the exceptions notedbelow) can be used at greater than its usual, recommended concentration,for example at 2×, 3×, 4×, 5×, 6×, 7×, 8×, or even higherconcentrations. As used herein, “1×” means the standard concentration,“2×” means twice the standard concentration, etc. In any of theseembodiments, medium components that can substantially affect osmolarity,such as salts, cannot be increased in concentration so that theosmolarity of the medium falls outside of an acceptable range. Thus, amedium may, for example, be 8× with respect to all components exceptsalts, which can be present at only 1×. An enriched medium may be serumfree and/or protein free. Further, a medium may be supplementedperiodically during the time a culture is maintained to replenish mediumcomponents that can become depleted such as, for example, vitamins,amino acids, and metabolic precursors. As is known in the art, differentmedia and temperatures may have somewhat different effects on differentcell lines, and the same medium and temperature may not be suitable forall cell lines.

Suitable culture conditions for mammalian cells are known in the art.See e.g. Animal cell culture: A Practical Approach, D. Rickwood, ed.,Oxford university press, New York (1992). Mammalian cells may becultured in suspension or while attached to a solid substrate.Furthermore, mammalian cells may be cultured, for example, in fluidizedbed bioreactors, hollow fiber bioreactors, roller bottles, shake flasks,or stirred tank bioreactors or any other bioreactor described herein orknown in the art, with or without microcarriers, and operated in abatch, fed batch, continuous, semi-continuous, or perfusion mode.

Monitoring Culture Conditions

In certain embodiments of the present invention, the practitioner mayfind it beneficial or necessary to periodically monitor particularconditions of the growing cell culture. Monitoring cell cultureconditions allows the practitioner to determine whether the cell cultureis producing recombinant polypeptide or protein at suboptimal levels orwhether the culture is about to enter into a suboptimal productionphase. In order to monitor certain cell culture conditions, it will benecessary to remove small aliquots of the culture for analysis. One ofordinary skill in the art will understand that such removal maypotentially introduce contamination into the cell culture, and will takeappropriate care to minimize the risk of such contamination.

As non-limiting example, it may be beneficial or necessary to monitortemperature, pH, cell density, cell viability, integrated viable celldensity, lactate levels, ammonium levels, osmolarity, or titer of theexpressed polypeptide or protein. Numerous techniques are well known inthe art that will allow one of ordinary skill in the art to measurethese conditions. For example, cell density may be measured using ahemacytometer, a Coulter counter, or Cell density examination (CEDEX).Viable cell density may be determined by staining a culture sample withTrypan blue. Since only dead cells take up the Trypan blue, viable celldensity can be determined by counting the total number of cells,dividing the number of cells that take up the dye by the total number ofcells, and taking the reciprocal. Cell viability and/or cell density canalso be measured using a biomass capacitance probe. HPLC, UPLC, NOVAFlex or Cedex Bio can be used to determine the levels of lactate,ammonium or the expressed polypeptide or protein. Alternatively, thelevel of the expressed polypeptide or protein can be determined bystandard molecular biology techniques such as coomassie staining ofSDS-PAGE gels, Western blotting, Bradford assays, Lowry assays, Biuretassays, and UV absorbance. It may also be beneficial or necessary tomonitor the post-translational modifications of the expressedpolypeptide or protein, including phosphorylation and glycosylation.

EXAMPLES Example 1

The following example describes the use of a biomass capacitance probeto precisely control cell growth as measured through the capacitanceprobe by modulating reactor temperature.

Cell Line and Cell Culture Medium

A BMS-proprietary cell culture medium and a proprietary recombinant CHOcell line expressing IgG antibodies were used for the experiments. Thecell culture medium was chemically defined and fully serum and proteinfree. Recombinant IgG producing CHO cells were maintained in suspensionculture in 250-mL, 1-L and 3 L shake flasks. A CO₂ shaker incubator(Kuhner) was used for incubation at 36.5° C., 150 rpm at a CO₂concentration of 5%.

Bench-Style Bioreactors and Cultivation Conditions

Bench scale reactor cultivations were carried out in 5-L stirred tankreactors (Sartorius). All the 5-L reactors were equipped with two 45°pitched tri-blade impellers, pH, DO (dissolved oxygen) and biomasscapacitance probes. The working volume of the 5-L bioreactor was 4.5 L.pH was controlled in the range of 7.1 to 7.4 by the addition of 1Msodium carbonate and CO₂ gas sparging. The impeller agitation was set at290 rpm, and aeration was provided by pure oxygen sparging through 0.5mm drilled hole spargers. Dissolved oxygen was maintained at a level of40% via cascade oxygen sparging. Antifoam (EX-CELL antifoam,Sigma-Aldrich) was added to the bioreactor to control foam levels. Thecells were inoculated at a density of 6×10⁵ cells/ml. Cell density wasmeasured by daily offline measurements (Vi-cell, Beckman Coulter) and/orvia online capacitance probes (Hamilton). Daily offline samples werealso monitored for pH, dissolved oxygen and pCO₂ via pHOx instruments(Nova Biomedical) and glucose, lactate profiles were measured with CedexBio HT (Roche). After centrifugation for 5 min at 1000 g, cell-freesamples were frozen at −80° C. before measurement of IgG titer by UPLCmethods.

Two feeding strategies were employed: daily and hourly bolus feeding.Daily bolus feeds of chemically defined media were added to theproduction vessel starting from day 3. The daily feed volume was set to3.6% of initial working volume. For hourly feeds, the normal daily bolusamount was divided by 24 (0.15% of initial working volume). This hourlyamount of feed was added to the reactor hourly starting at day 2.5.Strategy for control of cell growth in production bioreactors

The control strategy is diagramed in FIG. 1. The biomass was measuredusing a Hamilton Incyte capacitance biomass probe. The probe sends acontinuous biomass signal to the DeltaV control system, which controlsthe bioreactors. A pre-determined viable cell density was programmedinto DeltaV using Equation 1, and the constants and variables in Table 1were used.

$\begin{matrix}{{{Cell}\mspace{14mu} {Density}} = {\frac{A}{B*e^{({C*{({t - D})}})}} - e^{E}}} & {{Equation}\mspace{14mu} 1}\end{matrix}$

Active control was used from the time of inoculation (t=0) until peakviable cell density (t=240 h). The shape of Equation 1 over that timeframe is shown in FIG. 2.

The biomass measured from the capacitance probe was controlled using aproportional-integral-derivative (PID) controller with the viable celldensity from Equation 1 as the set-point. The PID values for the twofeed strategies are found in Table 2.

TABLE 2 PID settings Strategy Daily Hourly P (gain) 2 1 I (s) 400 60 D(s) 600 0 Filter (s) 300 300

The set point for the reactor temperature controller was cascaded fromthe biomass control loop described above. The biomass output wastranslated linearly to a temperature range of 31−37° C. The controllermaintained this set-point temperature through a heating blanket attachedto the reactor.

Cell Growth with Traditional Temperature Settings

As a control, three reactor cultures were grown using a standardtemperature shift. In these reactors, the temperature was maintained at36.5° C. for the first five days of culture, at which point thetemperature was lowered to 35.5, 34.0, or 32.5° C., as can be seen inFIG. 3.

Two PID control strategies were used as described in Table 2. Thetemperature and biomass profiles are presented in FIG. 4 for the dailyfeed, and in FIG. 5 for the hourly feed.

The above experiments were designed to precisely control cell growth asmeasured through an online capacitance probe by modulating reactortemperature. To achieve these results reactors were equipped with anonline dielectric capacitance probe which uses permittivity to measurebiomass density. To avoid major perturbations near continuous feedingstrategies were used. Biomass measurements from the probe were comparedto a pre-determined growth curve selected by the experiment operator. Atemperature control loop was then used to adjust the growth rate of thecells to match the pre-determined growth curve. This allowed precisecontrol of the timing of cell growth from inoculation to peak.

These experiments demonstrated it is possible to precisely controlbiomass growth in a reactor using a feedback loop set up with an onlinecapacitance probe. Two strategies were presented. The first strategyutilized a standard daily feed schedule as shown in FIG. 4. Biomass wasstill controlled using this strategy, but deviations from the targetline occurred after feeding events. The biomass control loop moved tocorrect these deviations, resulting in daily temperature oscillationswith a peak to peak amplitude of approximately 2° C.

Alternatively, an hourly feed schedule was utilized in the secondstrategy. By feeding the culture on an hourly basis, large biomassfluctuations were minimized. Biomass control was achieved with minimaldeviations from the target. The temperature profile was also lessvolatile, and did not have any periodic oscillations.

Identical bioreactors can also be controlled to peak at distinct timesseparated by a full day, or at distinct densities. These resultsdemonstrate new opportunities to improve mammalian process performancethrough more precisely controlled experimental designs and increasedrobustness in the face of process variability.

1. A method of controlling the growth of cells in a bioreactorcomprising: a) measuring the viable cell density of the cells growing inculture using a biomass capacitance probe; and b) utilizing atemperature control loop to adjust the growth rate of the cells.
 2. Amethod of modulating reactor temperature to control the growth of cellsin a bioreactor comprising: a) measuring the viable cell density of thecells growing in culture using a biomass capacitance probe; and b)utilizing a temperature control loop to modulate the reactor temperatureand adjust the growth rate of the cells.
 3. A method of minimizing thegrowth fluctuation of cells in a bioreactor comprising: a) measuring theviable cell density of cells growing in culture using a biomasscapacitance probe; and b) utilizing a temperature control loop to adjustthe growth rate of the cells.
 4. The method of claim 1, wherein apre-determined growth curve is utilized to adjust the temperature in theculture.
 5. The method of claim 1, wherein a bolus feed is added to thebioreactor daily.
 6. The method of claim 5, wherein the daily bolus feedis begun starting on about day 1, about day 1.5, about day 2, about day2.5, about day 3, about day 3.5, about day 4, about day 5, about day 6,about day 7, about day 8, about day 9 or about day 10 after the cultureis inoculated.
 7. The method of claim 5, wherein the daily bolus feedvolume is about 1%, about 2%, about 3%, about 4%, about 5%, about 6%,about 7%, about 8%, about 9%, about 10%, about 15% or more of theinitial working volume.
 8. The method of claim 5, wherein the dailybolus feed volume is about 3%, about 3.1%, about 3.2%, about 3.3%, about3.4%, about 3.5%, about 3.6%, about 3.7%, about 3.8, about 3.9% or about4% of the initial working volume.
 9. The method of claim 5, wherein thedaily bolus feed volume is about 3.6% of the initial working volume. 10.The method of claim 1, wherein a bolus feed is added to the bioreactorhourly.
 11. The method of claim 10, wherein the hourly bolus feed isbegun starting on about day 1, about day 1.5, about day 2, about day2.5, about day 3, about day 3.5, about day 4, about day 5, about day 6,about day 7, about day 8, about day 9 or about day 10 after the cultureis inoculated.
 12. The method of claim 10, wherein the hourly bolus feedis about 1/24 of the calculated volume for a daily bolus feed.
 13. Themethod of claim 10, wherein the hourly bolus feed is about 0.1%, about0.11%, about 0.12%, about 0.13%, about 0.14%, about 0.15%, about 0.16%,about 0.17%, about 0.18%, about 0.19%, about 0.2%, about 0.25%, about0.3%, about 0.4% or about 0.5% of the initial working volume.
 14. Themethod of claim 10, wherein the hourly bolus feed volume is about 0.15%of the initial working volume.
 15. The method of claim 1, wherein theviable cell density is plotted to a temperature range of about 30-40° C.16. The method of claim 1, wherein the viable cell density is plotted toa temperature range of about 31-37° C.
 17. The method of claim 1,wherein daily temperature oscillations in the reactor have a peak topeak amplitude of about 10° C., about 9° C., about 8° C., about 7° C.,about 6° C., about 5° C., about 4° C., about 3° C., about 2° C., about1° C. or less.
 18. The method of claim 1, wherein the cells produce apolypeptide of interest.
 19. The method of claim 18, wherein thepolypeptide of interest is an antibody.
 20. The method of claim 1,wherein the cells are mammalian cells.
 21. The method of claim 1,wherein the cells are Chinese Hamster Ovary (CHO) cells.
 22. The methodof claim 1, wherein the biomass capacitance probe is an INCYTE probe.23. The method of claim 1, wherein the pH of the culture is about 6.5 toabout 8.0, about 7.0 to about 8.0, or about 7.0 to about 7.5.
 24. Themethod of claim 1, wherein the pH of the culture is about 7.1 to about7.4.
 25. The method of claim 1, wherein the culture is inoculated withcells at a density of about 1×10⁵, about 2×10⁵, about 3×10⁵, about4×10⁵, about 5×10⁵, about 6×10⁵, about 7×10⁵, about 8×10⁵, about 9×10⁵,or about 10×10⁵ cells/ml.
 26. The method of claim 1, wherein the cultureis inoculated with cells at a density of about 6×10⁵ cells/ml.